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Abstract

The role of tumor cell expression of major histocompatibility class II (MHCII) has been controversial, with evidence indicating that tumor cell expression of MHCII may lead to an anti‑tumor immune response and to tumor cell apoptosis and that MHCII has pro‑tumorigenic functions. The cancer genome atlas (TCGA) indicates numerous deleterious mutations for the highly specific, MHCII transcriptional activation proteins, RFX5, RFXAP, RFXANK and CIITA. Also, mutations in the non‑polymorphic, human leukocyte antigen (HLA)‑DRA gene, which encodes the heavy chain for the most prominent human MHCII molecule, HLA‑DR, are common. For many, if not most TCGA cancer datasets, the MHCII specific mutations do not associate with clinical outcomes. However, stomach carcinoma represents an exception, where the data indicate that MHCII‑specific mutations are associated with a more favorable outcome. These data raise the question of whether stomach cancer mutations represent effective haploinsufficiency or whether mutations that are associated with a favorable outcome occur with other stomach cancer molecular features that limit the function of the two alleles that represent these MHCII‑related proteins.

Introduction

The potential impact of MHCII expression on solid
tumor cells received increased interest when it became apparent
over two decades ago that mutations specific to tumorigenesis
interfered with MHCII induction by interferon-γ (IFN-γ) (1–9). As one
example, a lack of retinoblastoma tumor suppressor protein leads to
over expression of the pro-proliferative protein, YY1, which in
turn is part of a repressive complex that maintains histone
deacetylase activity at the MHCII promoter, thereby blocking the
assembly of MHCII enhanceosome proteins, including the highly
specific MHCII enhanceosome proteins, RFXANK, RFXAP, RFX5 and CIITA
(10,11). In addition, Ostrand-Rosenberg and
colleagues (12,13) have established the negative impact of
tumor cell-MHCII expression on tumor development, although there
remain questions about whether such a negative impact occurs in a
natural state, where there is the expectation of CLIP expression
blocking endogenous MHCII tumor-peptide loading, or in the absence
of tumor cell expression of conventional co-stimulatory molecules.
The apoptotic mechanisms of tumor cell MHCII expression provide
another possible ‘anti-tumor’ role (14,15).

Data collection methods

Clinical and primary tumor specimen mutation
Microsoft Excel files for the stomach adenocarcinoma (STAD), skin
cutaneous melanoma (SKCM), lung adenocarcinoma (LUAD), colon
adenocarcinoma (COAD), head and neck squamous cell carcinoma (HNSC)
and bladder urothelial carcinoma (BLCA) cancer sets was downloaded
from the TCGA data portal (dbGaP project approval number 6300). The
‘new tumor event after initial treatment’ column of the TCGA
clinical follow up file for each cancer dataset was used to
categorize barcodes based on the development of a new tumor or not
(Table I, New Tumor and No
Subsequent Tumor, respectively). To obtain matching barcodes, for
the clinical and somatic mutation files, the barcodes from the
primary tumor specimen mutation file were truncated to contain the
following characters, TCGA-##-####. Mutation data, including
truncated tumor sample barcodes, human genome organization symbols
and mutation type (nonsynonymous or silent) for HLA-DRA and the set
of transcription factors (CIITA, RFX5, RFXANK and RFXAP) associated
with MHC Class II were collected for each cancer dataset. Mutations
were assessed using the PROVEAN web tool (16) The Excel COUNTIF function was used to
obtain the number of MHC Class II coding region mutations per
barcode for the New Tumor and No Subsequent Tumor groups for each
cancer dataset and a statistical comparison between the groups was
conducted. t-tests were used to obtain P-values. P<0.05 was
considered to indicate a statistically significant difference.
P-values were obtained using Microsoft Excel (Microsoft
Corporation, Redmond, WA, USA).

All procedures performed in studies involving human
participants were in accordance with the ethical standards of the
institutional and national research committee and with the 1964
Helsinki declaration and its later amendments or comparable ethical
standards. The present study is exempt from IRB approval and was
approved via the National Institutes of Health, Database of
Phenotypes and Genotypes (dbGaP), project no. 6,300; approval
granted to George Blanck.

Results and discussion

TCGA provides a wealth of information regarding
mutagenesis in many cancer datasets. To obtain an indication of
mutations that may impact MHCII expression, TCGA was searched for
mutations in RFXAP, RFXANK, RFX5, CIITA and HLA-DRA. Other MHCII
structural genes were excluded due to the potential confusion
caused by the high level of polymorphisms. Overall, the
nonsynonymous mutation rate for the following TCGA datasets, for
the above collection of MHCII specific proteins, was ~8–9%: STAD,
SKCM, LUAD, COAD, HNSC and BLCA.

The opportunities for linking TCGA clinical
information, particularly negative vs. positive outcomes, to
particular mutations remains limited, largely owing to the minimal
overlap of barcodes (patient samples) for mutation results and
clinical information. However, it is possible to attempt to
correlate mutation results with either no-subsequent tumor or
new-tumor for the above cancer datasets, particularly due to the
relatively high number of barcodes available representing this
distinction (Table I). Of the six
TCGA datasets representing a substantial number of mutations and
no-subsequent tumor and new-tumor cases, only the stomach cancer
datasets (STAD) demonstrated a correlation with the MHCII specific
mutations, namely an association of more mutations with
no-subsequent tumor (Table II). Of
thirty-one mutations that were in the STAD no-subsequent tumor
group, 21 were assessable by the PROVEAN (17) web tool, of which 9 were deleterious
and 12 were neutral. The two mutations that were in the new-tumor
group were assessable by PROVEAN, revealing that 1 was deleterious
and 1 was neutral.

Table II.

The average number of mutations per
barcode and statistical comparison of the New Tumor and No
Subsequent Tumor sets

Table II.

The average number of mutations per
barcode and statistical comparison of the New Tumor and No
Subsequent Tumor sets

As aforementioned, solid tumor cell expression of
MHCII has led to contradictory impressions as to whether MHCII
facilitates or inhibits solid tumor development. The above data
support the former possibility, but no doubt there are a number of
circumstances in which the impact of solid tumor expression of
MHCII may have varying effects on tumor progression. For example,
varied solid tumors may be affected differently by constitutive
MHCII expression or MHCII induction by IFN-γ. Furthermore, MHCII
expression may have different impacts at different stages of
tumorigenesis. The issue of the variable impacts of immune function
spans the consideration of the role of the immune system in tumor
development. Immune checkpoint inhibitors have had great positive
benefits for at least a subset of patients (18), yet in other settings, evidence
indicates that inflammation, particularly chronic inflammation, is
associated with tumor development (19,20).

The negative impact of MHCII expression on solid
tumor cells may include induction of T-cell anergy (21), due to lack of costimulatory
molecules, but a previous study indicates that non-professional
antigen presenting cells, including solid tumor cells, are able to
employ substitute co-stimulatory molecules such as ICAM1 (22). Another potential explanation for a
negative impact of MHCII expression is the possibility that MHCII
facilitates T-cell engulfment by solid tumor cells (23,24).

In conclusion, the current study indicates that, at
least in certain situations, the expression of MHCII on tumor cells
may represent a negative prognosis. Such a conclusion calls into
question scenarios where MHCII-based interactions with the immune
system would facilitate an anti-tumor immune response.

Acknowledgements

The authors would like to acknowledge the support of
the Anna Valentine Program and the taxpayers of the State of
Florida.